Borrelia burgdorferi, a causative agent of Lyme disease, establishes persistent infection that can affect the joints, heart, skin, and nervous system. The abilities of this spirochete to disseminate and persist in a variety of sites in the mammalian host indicate that interactions with mammalian cells and extracellular matrix occur continually during infection. In fact, many documented or putative adhesins have been identified, suggesting that the concerted action of diverse adhesion pathways enables B. burgdorferi to negotiate successive steps during its characteristic multiphasic and multisystemic infection. Unfortunately, an important gap in our current knowledge is the lack of any mechanistic information to inform this attractive model. This paucity of insight is due in part to the fact that some of the adhesins identified to date display an overlap in binding activities, a factor that confounds straightforward analysis of any single adhesion pathway. Here we propose a comprehensive in vitro and in vivo analysis of the properties and function of each adhesin in the absence of potentially redundant mechanisms. To this end, this application brings together three principal investigators, each with a unique area of expertise, to address this critical knowledge gap. The three laboratories will employ state of the art approaches, including generation of gain-of-function B. burgdorferi mutants in a non-adherent, high-passage strain background and testing of the strains for in vitro adhesion activities (Aim 1, Leong lab), quantification of bacterial burdens for all gain-of-function mutants in comparison to the parental strain in mouse tissues (Aim 2, Coburn lab), and intravital microscopic examination of the nature of the interaction between each of the strains and the vasculature in living mice (Aim 3, Chaconas lab). This project therefore constitutes the first systematic examination of the roles of the diverse adhesins of B. burgdorferi in the ability of the organism to establish disseminated infection in immunocompetent animals. Our long-term goal is to understand how the biochemical activities of different adhesins function together to allow B. burgdorferi to overcome host barriers to the establishment of persistent, disseminated infection. As Lyme disease prevalence continues to expand in the Northern hemisphere, in terms of both case numbers and geographic distribution, this work may illuminate how B. burgdorferi causes Lyme disease in humans and persists in its animal reservoirs. This innovative multiple PI project brings together teams with unique expertise that will allow comprehensive investigations of a large set of B. burgdorferi proteins with defined biochemical activities. These proteins may be excellent candidates for development as vaccine candidates or for targeting as therapeutic interventions.